The cell division cycle is one of the most fundamental biological processes, which ensures the controlled production of cells with specialised functions. The progression through the eukaryotic cell cycle is controlled by the sequential formation, activation and inactivation of a whole series of protein-serine/threonine kinases, so-called cycline-dependent kinases (CDKs; cycline-dependent kinases) (cf. M. Peter et al. in Cell 79, 181-184 (1994) and G. F. Draetta in Cell Biology 6, 842-846 (1994)). Each CDK obviously phosphorylates specific substrates and coordinates the changes which occur during a specific transition status of the cell cycle. Because of the central role of the CDKs, vigorous attempts have been made to clarify and understand their regulation. In the mean time, some mechanisms have been identified. Thus, essential subunits are only accessible during the corresponding period of the cell cycle because of the synthesis and breakdown control of cycline. Moreover, some CDK/cycline complexes are inhibited by the binding of small protein inhibitors (cycline-dependent kinase inhibitors), whose presence is also strictly controlled. Furthermore, the CDK activities are translationally regulated by reversible phosphorylation of their catalytic subunits (cf. C. Hutchinson and D. M. Glover (Editors) in Cell Cycle Control, IRL Press, London, 1994).
The primary regulator of the CDK activity is the associated cycline subunit. Cyclines, which were originally defined as proteins, the concentrations of which oscillate during the cell cycle, are now--more accurately--defined as a family of structurally related proteins which bind and activate CDK-catalytic subunits. For example, CDK1 interacts with cycline B and with cycline A; CDK2 with cycline E and cycline A; CDK4 and CDK6 with D-cyclines; and CDK7 with cycline H. The cycline function is primarily controlled by changes in the cycline concentrations which increase characteristically when the cell is in a certain state: cycline E during the G1/S phase; cycline A during the S phase; cycline B during the G2/M phase. D-cyclines and cycline H are exceptions in this respect as their concentrations are relatively constant during the entire cell cycle.
The D-cyclines and cycline E and cycline A are primarily responsible for the progression through the G1 to the S phase of the cell cycle. Growth hormones, steroid hormones, the activation of ras, and other mitogenic stimuli induce an increase in the concentration of D-cyclines and/or cycline E and thereby initiate the progression of the cell through the G1 to the S phase. A substrate for cycline D/CDK4 or cycline D/CDK6 is the retinoblastoma gene product (pRB). The retinoblastoma gene in turn is a tumour suppressor gene which controls the cell proliferation. pRB--in the hypophosphorylated form--is normally bound to the transcription factor E.sub.2 F, which is inactive in this complex. Hyerphosphorylation of pRB by CDKs releases E.sub.2 F and induces transcription. A key role in cell growth is played by the cycline D/CDK4 or/CDK6 complex. There are increasingly indications that D-cyclines (D1 and D2) are obviously highly involved in the genesis of tumours (cf. L. H. Hartwell et al. in Science 266, 1821-1828 (1994)). The molecular mechanisms which underlie the proto-oncogenic properties of cycline D1 include chromosomal rearrangements (in parathyroid adenoma and B-cell lymphoma) and amplification of the chromosomal band 11q13, which has been reported for various types of cancer (including breast, head, neck and liver tumours) (cf. C. Gillett et al. in Cancer Research 54, 1812-1817 (1994) and T. Callender et al. in Cancer 74, 152-158 (1994)). It is assumed that the overall result of these genetic changes is an ectopic or abnormally heightened expression of the cycline D1 protein, which may possibly contribute to excessive cell divisions and unregulated tumour growth.
Another major mechanism of CDK regulation involves a family of different proteins, so-called cycline-dependent kinase inhibitors (CKIs) which bind and inhibit cycline/CDK complexes (cf. G. Peters in Nature 371, 204-205 (1994)). The chief (mammalian) CKIs fall into two categories: (1): p21 (CIP1/WAF1/-CAP20/SD1), p27 (KIP1) and p57 (KIP2) are related proteins with a preference for cycline/CDK2 and cycline/CDK4 complexes; (2) p16.sup.INK4, p15.sup.INK4B, p18.sup.INK4C and p19.sup.INK4D are closely related CKIs with a specificity for CDK4 and/or CDK6. p21 primarily regulates transcription. p21 transcription is induced by the tumour-suppressor gene p53, a transcriptional regulator which mediates the stopping of the cell cycle after DNA damage or in senescence. Basal concentrations of p21 may possibly constitute a threshold which has to be crossed before complexes can become active. Transcriptional control may possibly also be important for p15.sup.INK4B, the expression of which is greatly increased when treated with the negative growth factor TGF.beta.. An additional effect of TGF.beta. is obviously the release of p27, which is established in a heat-sensitive compartment. p27 is probably also involved in the effects of positive growth factors. For example, interleukin-2 stimulation appears to induce a fall in the concentration of p27 and as a result the proliferation of T-cells.
Very recent studies have frequently shown an allelic loss at chromosome 9 in a number of human carcinomas (e.g. melanoma, head and neck squamous cell cancer, lung cancer, pancreatic adeno-Ca, breast cancer and nasopharyngeal-Ca) (cf. A. Kamb et al. in Science 264, 436-440 (1994); C. J. Hussussian et al. in Nature Genetics 8, 15-21 (1994); C. Caldas et al. in Cancer Nature Genetics 8, 27-32 (1994); T. Mori et al. in Cancer Research 54, 3396-3397 (1994) and A. Okamato et al. in Proc. Natl. Acad. Sci. USA 91, 11045-11049 (1994)). The loss of chromosome 9p21-22 is of particular interest. In this region, where a tumour suppressor gene is presumed to be, there is a gene bearing the name CDKN2 (MTS 1, Multiple Tumour Suppressor Gene 1), which codes a p16 protein. As already mentioned above, the p16 protein binds to CDK4 and CDK6 and thus inhibits their interaction with D-cyclines. Damage or mutations in the p16 gene may possibly influence the relative balance of functional p16 and cycline D, leading to unregulated CDK activity and abnormal cell growth. The very recent observations that p16 damage, inactivation by gene silencing and/or mutations very frequently occur in many tumour cells, indicate that p16 plays a key role in suppressing the development of various human carcinomas (cf. G. I. Shapiro et al. in Cancer Research 55, 6200-6209 (1995)).
Deregulated CDK activity may also be the consequence of: (a) mutation or excessive expression of the kinase; (b) induced expression, overexpression or delayed breakdown of cyclines; (c) functional inactivation of CKIs by gene silencing, damage or mutation; or (d) a combination of these phenomena. The result of these deviations is a deregulated cell cycle with deregulated cell division, which causes various illnesses or contributes to their progress.